Hemostasis is a natural process by which injured blood vessels are repaired through the combined activity of vascular, platelet, and plasma factors, counterbalanced by regulatory mechanisms which control the amount of activated platelets and fibrin at the site of injury. Vascular factors reduce blood flow to the injured blood vessels by local vasoconstriction and by compression of the blood vessels due to blood flow into the surrounding tissues. Hemostatic plugs to seal the damaged blood vessels are formed by activation of platelet adhesion followed by blood coagulation reactions leading to the formation of a fibrin clot. Hemostatic abnormalities can result in excessive bleeding or thrombosis. Activation of this coagulation cascade may contribute to inflammation.
In the blood coagulation pathway, serine protease proenzymes are activated, resulting in the formation of a prothrombin activator which is a complex of the enzyme Factor Xa, and cofactors Va and procoagulant phospholipid (anionic phospholipid present in plasma membranes of platelets, endothelial cells and erythrocytes). The prothrombin activator splits prothrombin into two parts, one of which is the enzyme thrombin. Thrombin then reacts with fibrinogen to form fibrin and also activates Factor XIII, an enzyme which catalyzes the formation of covalent bonds between fibrin molecules, resulting in the formation of a clot. Impairment of the blood coagulation pathway leads to excessive bleeding tendencies.
Regulatory mechanisms normally control activated coagulation, thus preventing the spread of local thrombosis or disseminated intravascular coagulation. One method of regulation involves the neutralization of enzymes and activated cofactors in the blood which are necessary for coagulation, e.g., Factors Va and VIIIa. In this process, thrombin binds to a receptor on the endothelial cell membrane called thrombomodulin. When bound to thrombomodulin, thrombin loses its ability to convert fibrinogen to fibrin and activates Protein C, a vitamin K-dependent serine protease enzyme. In the presence of Protein S (also a vitamin K-dependent protein) and phospholipid, activated Protein C catalyzes the proteolysis of Factors VIIIa and Va, destroying their cofactor function and subsequently terminating clot formation. Failure to activate Protein C may result in numerous thromboembolic manifestations and increased inflammation. Additionally, studies in the baboon Papio cynocephalus cynocephalus indicate that the Protein C pathway is critical in modulating the inflammatory and coagulopathic responses in vivo. (Taylor, et al., "Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon, " J Clin Invest 79:918-925 (1987)) Inhibition of Protein C activation by the thrombin-thrombomodulin complex leads to disruption of this regulatory pathway and uncontrolled coagulation, thrombosis, and inflammation. This concept is supported by the observation that patients with Protein C deficiency demonstrate thrombotic complications which are corrected by Protein C replacement therapy. (Esmon, "The regulation of natural anticoagulant pathways," Science 235:1348-1352 (1987); Dreyfus, et al., "Treatment of homozygous Protein C deficiency and neonatal purpura fulminans with a purified Protein C concentrate," New Eng J of Med 325:1565-1568 (1991))
Endogenous substances such as circulating anticoagulants (e.g., lupus coagulant) may affect blood coagulation. Lupus anticoagulant is a circulating anticoagulant which was first described in patients with systemic lupus erythematosus (SLE) and subsequently found associated with a wide variety of disorders. It is characterized as an immunoglobulin, or antibody, which reacts with anionic phospholipids and/or coagulation factors such as prothrombin used in in vitro coagulation assays such as PTT (partial thromboplastin time) and aPTT (activated partial thromboplastin time). The presence of lupus anticoagulant in the patient's plasma results in a prolonged PTT or aPTT which fails to correct with a 1:1 mixture of the patient's plasma and normal plasma.
Antiphospholipid antibodies have been found in plasma from many patients with lupus anticoagulant using an ELISA assay looking for antibodies which bind cardiolipin (Triplett, et al., "The relationship between lupus anticoagulants and antibodies to phospholipid," JAMA 259:550-554 (1988)) possibly in association with coagulation proteins or .beta.-2-glycoprotein-1 (Pengo, et al., "Immunological specificity and mechanism of action of IgG lupus anticoagulants," Blood 70:69-76 (1987); Thiagarajan, et al., "Monoclonal immunoglobulin M: coagulation inhibitor with phospholipid specificity," J Clin Invest 66:397-405 (1980)).
Although the lupus anticoagulant inhibits the function of phospholipid and coagulation factors in in vitro coagulation assays, most patients with lupus anticoagulant do not bleed excessively, rather they have thrombosis. Paradoxically, patients with lupus anticoagulant demonstrate an increased risk of thrombosis, e.g., recurrent thromboembolism, myocardial infarction, stroke, thrombotic spontaneous abortion, and thrombocytopenia. However, not all patients with lupus anticoagulant have a propensity for thrombosis, and many patients suffering from unexplained thrombotic diseases do not test positively to lupus anticoagulant. Likewise, not all patients with SLE test positive for lupus anticoagulant and not all patients with lupus anticoagulant have SLE.
The pathogenesis of thrombosis associated with lupus anticoagulant has not been clearly elucidated. Extensive research has been conducted in an effort to explain the relationship between lupus anticoagulant and thrombotic episodes. Several investigators noted that antibodies from patients with lupus anticoagulant block the activation of Protein C by the thrombin-thrombomodulin complex. It has been proposed that these antibodies interfere with the phospholipid enhancement of Protein C activation by the thrombin-thrombomodulin complex. Cariou et al., "Inhibition of Protein C activation by endothelial cells in the presence of lupus anticoagulant," New England J of Med 314:1193-1194 (1986); Cariou et al., "Effect of lupus anticoagulant on antithrombogenic properties of endothelial cells-inhibition of thrombomodulin-dependent Protein C activation," Thrombosis and Haemostasis 60:54-58 (1988); Freyssinet, et al., "An IgM lupus anticoagulant that neutralizes the enhancing effect of phospholipid on purified endothelial thrombomodulin activity-a mechanism for thrombosis," Thrombosis and Haemostasis 55:309-313 (1986). This hypothesis is supported by the fact that added phospholipid neutralizes the ability of lupus anticoagulant immunoglobulin to inhibit thrombomodulin function. Cariou et al., "Effect of lupus anticoagulant on antithrombogenic properties of endothelial cells-inhibition of thrombomodulin-dependent Protein C activation," Thrombosis and Haemostasis 60:54-58 (1988); Freyssinet, et al., "The effect of phospholipids on the activation of protein C by the human thrombin-thrombomodulin complex," Biochem J 238:151-157 (1986); Freyssinet, et al., "An IgM lupus anticoagulant that neutralizes the enhancing effect of phospholipid on purified endothelial thrombomodulin activity--a mechanism for thrombosis," Thrombosis and Haemostasis 55:309-313 (1986). However, in some cases where antithrombomodulin activity has been identified in thrombomodulin-dependent Protein C activation assays, disparities have been found between the titer of antiphospholipid antibody and the amount of antithrombomodulin activity, suggesting that the mechanism of thrombosis may involve more than blocking phospholipids. Cariou, et al., "Effect of lupus anticoagulant on antithrombogenic properties of endothelial cells-inhibition of thrombomodulin-dependent protein C activation," Thrombosis and Haemostasis 60:54-58 (1988); Oosting, et al., "In vitro studies of antiphospholipid antibodies and its cofactor, .beta.-glycoprotein I, show negligible effects on endothelial cell mediated Protein C activation," Thrombosis and Haemostasis 66:666-671 (1991); Triplett, et al., "The laboratory heterogeneity of lupus anticoagulants," Arch Pathol Lab Med 109:946-951 (1985); Tsakiris, et al., "Lupus anticoagulant--antiphospholipid antibodies and thrombophilia. Relation to Protein C--Protein S--thrombomodulin," J Rheumatol 17:785-789 (1990); Ruiz-Arguelles, et al., "Acquired protein C deficiency in a patient with primary antiphospholipid syndrome. Relationship to reactivity of anticardiolipin antibody with thrombomodulin," J Rheumatol 16:381-383 (1989).
Gibson et al attempted to demonstrate the direct binding of antibodies to thrombomodulin as a possible mechanism for blocking the activation of Protein C by the thrombin-thrombomodulin complex. Plasma from patients with lupus anticoagulant was examined for the presence of antibodies to thrombomodulin using an enzyme-linked immunosorbent assay (ELISA). In this assay, purified human placental thrombomodulin was coated to wells of a microtiter plate which was then probed with patient plasma. The results indicated that there was no significant antibody reactivity to thrombomodulin in patients with lupus anticoagulant when compared to lupus anticoagulant negative patients. The conclusion of the study was that antibodies to thrombomodulin do not exist. Gibson, et al., "Autoantibodies to thrombomodulin: development of an enzyme immanunoassay and a survey of their frequency in patients with the lupus anticoagulant," Thrombosis and Haemostasis 67:507-509 (1992).
Natural human thrombomodulin is a 75,000 kD endothelial cell protein having a structure which resembles the low density lipoprotein (LDL) receptor with an amino terminal lectin-like region followed by six tandem epidermal growth factor (EGF)-like repeats. The last three EGF-like repeats (EGF 456) contain the region required for thrombin binding and Protein C activation. Zushi, et al., "The last three consecutive epidermal growth factor-like structures of human thrombomodulin comprise the minimum functional domain for protein C--activating cofactor activity and anticoagulant activity," J Biol Chem 264:10351-10353 (1989); Tsiang, et al., "Functional domains of membrane-bound human thrombomodulin. EGF 456 domains and the serine/threonine-rich domain are required for cofactor activity," J Biol Chem 267:6164-6170 (1992). After the EGF-like repeats is a serine/threonine-rich region containing chondroitin sulfate, followed by a transmembrane domain and a short cytoplasmic tail. The basic amino acid structure of human thrombomodulin cDNA (SEQ ID NO:1) is presented sequentially in Suzuki, et al., "Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation," EMBO J 6:1891 (1987): signal peptide (16, 18, or 21 residues), amino-terminal lectin domain (223-226 residues), six EGF-like (epidermal growth factor) domains (236-240 residues), serine-threonin rich chondroitin sulfate domain (34-37 residues), transmembrane region (23-24 residues), and cytoplasmic domain (36-38 residues), for a total of 575 amino acid residues.
Oosting, et al. identified patient immunoglobulin fractions (IgG) which inhibited Protein C activation and was able to demonstrate by ELISA binding of these antibodies to the epidermal growth factors (EGF) domain in SLE patients with thrombotic complications. (Oosting, "Autoantibodies directed against the epidermal growth factor-like domains of thrombomodulin inhibit protein C activation in vitro," British Journal of Haematology 85:761-768 (1993). These authors concluded that autoantibodies to thrombomodulin must be directed specifically against the restricted region of thrombomodulin containing the epidermal growth factor (EGF) domain.
In contrast to the reports of the literature, it has now been found that antibodies to various regions of thrombomodulin exist, and an assay for detection of these antibodies has been developed as a diagnostic tool for patients having a propensity for unexplained thrombosis or inflammation whether or not they have lupus anticoagulant.